Start Me Up: Endocytosis on demand

We have a new paper out. The title is New tools for ‘hot-wiring’ clathrin-mediated endocytosis with temporal and spatial precision. You can read it here.

Cells have a plasma membrane which is the barrier between the cell’s interior and the outside world. In order to import material from outside, cells have a special process called endocytosis. During endocytosis, cells form a tiny bubble of plasma membrane and pull it inside – taking with it a little pocket of the outside world. This process is very important to the cell. For example, it is one way that cells import nutrients to live. It also controls cell movement, growth, and how cells talk to one another. Because it is so important, cell biologists have studied how endocytosis works for decades.

Studying endocytosis is tricky. Like naughty children, cells simply do not do what they are told. There is no way to make a cell in the lab “do endocytosis”. It does it all the time, but we don’t know when or where on the cell surface a vesicle will be made. Not only that, but when a vesicle is made, we don’t really know what cargo it contains. It would be helpful to cell biologists if we could bring cells under control. This paper shows a way to do this. We demonstrate that clathrin-mediated endocytosis can be triggered, so that we can make it happen on-demand.

Endocytosis on-demand

Using a chemical which diffuses into the cell, we can trigger endocytosis to happen all over the cell. The movie on the right shows vesicles (bright white spots) forming after we add the chemical (at 0:00). The way that we designed the system means that the vesicles that form have one type of cargo in there. This is exciting because it means that we can now deliver things into cells using this cargo. So, we can trigger endocytosis on-demand and we can control the cargo, but we still cannot control where on the plasma membrane this happens.

We solved this problem by engineering a light-sensitive version of our system. With this new version we can use blue light to trigger endocytosis. Whereas the chemical diffused everywhere, the light can be focussed in a narrow region on the cell and endocytosis can be trigger only in that region. This means we control where, as well as when, a vesicle will form.

What does hot-wiring mean?

It is possible to start a car without a key by “hot-wiring” it. This happens in the movies, when the bad guy breaks into a car and just twists some wires together to start the car and make a getaway. To trigger endocytosis we used the cell’s own proteins, but we modified them. We chopped out all the unnecessary parts and just left the bare essentials. We call the process of triggering endocytosis “hot-wiring” because it is similar to just twisting the wires together rather than having a key.

It turns out that movies are not like real life, and hot-wiring a car is actually quite difficult and takes a while. So our systems are more like the Hollywood version than real life!

What is this useful for?

As mentioned above, the systems we have made are useful for cell biologists because they allow cells to be “tamed”. This means that we can accurately study the timing of endocytosis and which proteins are required in a very controlled way. It also potentially means that molecules can be delivered to cells that cannot normally enter. So we have a way to “force feed” cells with whatever we want. This would be most useful for drugs or nanoparticles that are not actively taken up by cells.

Who did the work?

Almost all of the work in the paper was by Laura Wood, a PhD student in the lab. She had help from fellow lab members Nick Clarke, who did the correlative light-electron microscopy, and Sourav Sarkar who did the binding experiments. Gabrielle Larocque, another PhD student did some fantastic work to revise the paper after Laura had departed for a post-doc position at another University. We put the paper up on bioRxiv in Summer 2016 and the paper has slowly made its way through peer review to be published in J Cell Biol today.

Wait? I’m a cell biologist! I want to know how this thing really works!

OK. The design is shown to the right. We made a plasma membrane “anchor” and a clathrin “hook” which is a fragment of protein which binds clathrin. The anchor and the hook have an FRB domain and an FKBP domain and these can be brought together by rapamycin. When the clathrin hook is at the membrane this is recognised by clathrin and vesicle formation can begin. The main hook we use is the appendage and hinge from the beta2 subunit of the AP2 complex.

Normally AP2, which has four subunits, needs to bind to PIP2 in the plasma membrane and undergo a conformational change to recognise a cargo molecule with a specific motif, only then can clathrin bind the beta2 appendage and hinge. By hot-wiring, we effectively remove all of those other proteins and all of those steps to just bring the clathrin binding bit to the membrane when we want. Being able to recreate endocytosis using such a minimalist system was a surprise. In vitro work from Dannhauser and Ungewickell had suggested this might be possible, but it really seems that the steps before clathrin engagement are not a precursor for endocytosis.

To make the light inducible version we used TULIPs (tunable light-controlled interacting proteins). So instead of FRB and FKBP we had a LOVpep and PDZ domain on the hook and anchor.

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The post title comes from “Start Me Up” by The Rolling Stones. Originally on Tattoo You, but perhaps better known for its use by Microsoft in their Windows 95 advertising campaign. I’ve finally broken a rule that I wouldn’t use mainstream song titles for posts on this blog.

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2 thoughts on “Start Me Up: Endocytosis on demand”

Really enjoyed reading this paper! I was particularly interested to see that hotwired endocytosis does not inhibit endogenous CME (Figure 5) . Do you think it’s feasible to adapt this to be used as a reversible chemical/opto-controlled CME inhibitor?

Thanks for the suggestion Dave, great idea! We are working on this at the moment. Watch this space. We couldn’t think of a way of using a modified form of this system to get inhibition so we’re doing something a bit different.